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Abstract:

The present invention provides adipose-derived stem cells and lattices. In
one aspect, the present invention provides a lipo-derived stem cell
substantially free of adipocytes and red blood cells and clonal
populations of connective tissue stem cells. The cells can be employed,
alone or within biologically-compatible compositions, to generate
differentiated tissues and structures, both in vivo and in vitro.
Additionally, the cells can be expanded and cultured to produce hormones
and to provide conditioned culture media for supporting the growth and
expansion of other cell populations. In another aspect, the present
invention provides a lipo-derived lattice substantially devoid of cells,
which includes extracellular matrix material from adipose tissue. The
lattice can be used as a substrate to facilitate the growth and
differentiation of cells, whether in vivo or in vitro, into anlagen or
even mature tissues or structures.

20. The lipo-derived lattice of any of claims 17-19, comprising a hormone.

21. The lipo-derived lattice of claim 20, wherein the hormone is selected
from the group of hormones consisting of cytokines and growth factors.

22. The lipo-derived lattice of any of claims 17-21, which is
substantially anhydrous.

23. The lipo-derived lattice of any of claims 17-22, which is lyophilized.

24. The lipo-derived lattice of any of claims 17-21, which is hydrated.

25. A kit comprising the lipo-derived lattice of any of claims 17-24 and
one or more components selected from the group of components consisting
of hydrating agents, cell culture substrates, cell culture media,
antibiotic compounds, and hormones.

26. A composition comprising a cell and the lipo-derived lattice of any of
claims 17-24.

27. A composition comprising the cell of any of claims 1-8 and a
biologically compatible lattice.

28. A composition comprising the population of any of claims 9-16 and a
biologically compatible lattice.

33. The composition of any of claims 29-32, wherein the polymeric material
is a hydrogel formed by crosslinking of a polymer suspension having the
cells dispersed therein.

34. The composition of any of claims 29-33, wherein the lattice further
comprises a hormone selected from the group of hormones consisting of
cytokines and growth factors.

35. The composition of any of claims 29-34, wherein the lattice is the
lipo-derived lattice of any of claims 17-24.

36. A method of obtaining a genetically-modified cell comprising exposing
the cell of any of claims 1-8 to a gene transfer vector comprising a
nucleic acid including a transgene, whereby the nucleic acid is
introduced into the cell under conditions whereby the transgene is
expressed within the cell.

37. The method of claim 36, wherein the transgene encodes a protein
conferring resistance to a toxin.

38. A method of delivering a transgene to an animal comprising (a)
obtaining a genetically-modified cell in accordance with claim 36 or 37
and (b) introducing the cell into the animal, such that the transgene is
expressed in vivo.

39. A method of differentiating the cell of any of claims 1-8 comprising
culturing the cell in a morphogenic medium under conditions sufficient
for the cell to differentiate.

41. The method of claim 39 or 40, wherein the morphogenic medium is an
adipogenic medium and the cell is monitored to identify adipogenic
differentiation.

42. The method of claim 39 or 40, wherein the morphogenic medium is a
chondrogenic medium and the cell is monitored to identify chondrogenic
differentiation.

43. The method of claim 39 or 40, wherein the morphogenic medium is an
embryonic or fetal medium and the cell is monitored to identify embryonic
or fetal phenotype.

44. The method of claim 39 or 40, wherein the morphogenic medium is a
myogenic medium and the cell is monitored to identify myogenic
differentiation.

45. The method of claim 39 or 40, wherein the morphogenic medium is an
osteogenic medium and the cell is monitored to identify osteogenic
differentiation.

46. The method of claim 39 or 40, wherein the morphogenic medium is a
stromal medium and the cell is monitored to identify stromal or
hematopoetic differentiation.

47. The method of any of claims 39-46, wherein the cell differentiates in
vitro.

48. The method of any of claims 39-46, wherein the cell differentiates in
vivo.

49. A method of producing hormones, comprising (a) culturing the cell of
claim 7 or 8 within a medium under conditions sufficient for the cell to
secrete the hormone into the medium and (b) isolating the hormone from
the medium.

50. A method of promoting the closure of a wound within a patient
comprising introducing the cell of claim 7 or 8 into the vicinity of a
wound under conditions sufficient for the cell to produce the hormone,
whereby the presence of the hormone promotes closure of the wound.

51. A method of promoting neovascularization within tissue, comprising
introducing the cell of claim 7 or 8 into the tissue under conditions
sufficient for the cell to produce the hormone, whereby the presence of
the hormone promotes neovascularization within the tissue.

52. The method of claim 51, wherein the tissue is within an animal.

53. The method of claim 51 or 52, wherein the tissue is a graft.

54. The method of any of claims 49-53, wherein the hormone is a growth
factor selected from the group of growth factor consisting of human
growth factor, nerve growth factor, vascular and endothelial cell growth
factor, and members of the TGFβ superfamily.

55. A method of conditioning culture medium comprising exposing a cell
culture medium to the cell of any of claims 1-7 under conditions
sufficient for the cell to condition the medium.

56. The method of claim 55, wherein the medium is separated from the cell
after it has been conditioned.

57. The method of any of claims 36-56, wherein the cell is within a
population of any of claims 9-16.

58. A conditioned culture medium produced in accordance with the method of
claim 55 or 56.

59. The conditioned culture medium of claim 58, which is substantially
free of a cell of any of claims 1-7.

60. A method of culturing a stem cell comprising maintaining a stem cell
in the conditioned medium of claim 58 or 59 under conditions for the stem
cell to remain viable.

61. The method of claim 60, which further comprises permitting successive
rounds of mitotic division of the stem cell to form an expanded
population of stem cells.

62. The method of claim 60 or 61, wherein the medium is substantially free
of the lipo-derived cells of any of claims 1-7.

63. The method of any of claims 60-62, wherein the medium contains
lipo-derived cells of any of claims 1-7.

64. The method of claim 63, wherein a stem cell and a lipo-derived cell
are in contact.

65. The method of any of claims 60-64, wherein a stem cell is a
hemopoietic stem cell.

66. A method of producing animal matter comprising maintaining the
composition of any of claims 18-26 under conditions sufficient for the
cells within the composition to expand and differentiate to form the
matter.

Description:

BACKGROUND OF THE INVENTION

[0001]In recent years, the identification of mesenchymal stem cells,
chiefly obtained from bone marrow, has led to advances in tissue regrowth
and differentiation. Such cells are pluripotent cells found in bone
marrow and periosteum, and they are capable of differentiating into
various mesenchymal or connective tissues. For example, such bone-marrow
derived stem cells can be induced to develop into myocytes upon exposure
to agents such as 5-azacytidine (Wakitani et al., Muscle Nerve, 18(12),
1417-26 (1995)). It has been suggested that such cells are useful for
repair of tissues such as cartilage, fat, and bone (see, e.g., U.S. Pat.
Nos. 5,908,784, 5,906,934, 5,827,740, 5,827,735), and that they also have
applications through genetic modification (see, e.g., U.S. Pat. No.
5,591,625). While the identification of such cells has led to advances in
tissue regrowth and differentiation, the use of such cells is hampered by
several technical hurdles. One drawback to the use of such cells is that
they are very rare (representing as few as 1/2,000,000 cells), making any
process for obtaining and isolating them difficult and costly. Of course,
bone marrow harvest is universally painful to the donor. Moreover, such
cells are difficult to culture without inducing differentiation, unless
specifically screened sera lots are used, adding further cost and labor
to the use of such stem cells. Thus, there is a need for a more readily
available source for pluripotent stem cells, particularly cells that can
be cultured without the requirement for costly prescreening of culture
materials.

[0002]Other advances in tissue engineering have shown that cells can be
grown in specially-defined cultures to produce three-dimensional
structures. Spacial definition typically is achieved by using various
acellular lattices or matrices to support and guide cell growth and
differentiation. While this technique is still in its infancy,
experiments in animal models have demonstrated that it is possible to
employ various acellular lattice materials to regenerate whole tissues
(see, e.g., Probst et al. BJU Int., 85(3), 362-7 (2000)). A suitable
lattice material is secreted extracellular matrix material isolated from
tumor cell lines (e.g., Engelbreth-Holm-Swarm tumor secreted
matrix--"matrigel"). This material contains type IV collagen and growth
factors, and provides an excellent substrate for cell growth (see, e.g.,
Vukicevic et al., Exp. Cell Res, 202(1), 1-8 (1992)). However, as this
material also facilitates the malignant transformation of some cells
(see, e.g., Fridman, et al., Int. J. Cancer, 51(5), 740-44 (1992)), it is
not suitable for clinical application. While other artificial lattices
have been developed, these can prove toxic either to cells or to patients
when used in vivo. Accordingly, there remains a need for a lattice
material suitable for use as a substrate in culturing and growing
populations of cells.

BRIEF SUMMARY OF THE INVENTION

[0003]The present invention provides adipose-derived stem cells and
lattices. In one aspect, the present invention provides a lipo-derived
stem cell substantially free of adipocytes and red blood cells and clonal
populations of connective tissue stem cells. The cells can be employed,
alone or within biologically-compatible compositions, to generate
differentiated tissues and structures, both in vivo and in vitro.
Additionally, the cells can be expanded and cultured to produce hormones
and to provide conditioned culture media for supporting the growth and
expansion of other cell populations. In another aspect, the present
invention provides a lipo-derived lattice substantially devoid of cells,
which includes extracellular matrix material from adipose tissue. The
lattice can be used as a substrate to facilitate the growth and
differentiation of cells, whether in vivo or in vitro, into anlagen or
even mature tissues or structures.

[0004]Considering how plentiful adipose tissue is, the inventive cells and
lattice represent a ready source of pluripotent stem cells. Moreover,
because the cells can be passaged in culture in an undifferentiated state
under culture conditions not requiring prescreened lots of serum, the
inventive cells can be maintained with considerably less expense than
other types of stem cells. These and other advantages of the present
invention, as well as additional inventive features, will be apparent
from the accompanying drawings and in the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0005]One aspect of the invention pertains to a lipo-derived stem cell.
Preferably, the stem cell is substantially free of other cell types
(e.g., adipocytes, red blood cells, other stromal cells, etc.) and
extracellular matrix material; more preferably, the stem cell is
completely free of such other cell types and matrix material. Preferably,
the inventive cell is derived from the adipose tissue of a primate, and
more preferably a higher primate (e.g., a baboon or ape). Typically, the
inventive cell will be derived from human adipose tissue, using methods
such as described herein.

[0006]While the inventive cell can be any type of stem cell, for use in
tissue engineering, desirably the cell is of mesodermal origin. Typically
such cells, when isolated, retain two or more mesodermal or mesenchymal
developmental phenotypes (i.e., they are pluripotent). In particular,
such cells generally have the capacity to develop into mesodermal
tissues, such as mature adipose tissue, bone, various tissues of the
heart (e.g., pericardium, epicardium, epimyocardium, myocardium,
pericardium, valve tissue, etc.), dermal connective tissue, hemangial
tissues (e.g., corpuscles, endocardium, vascular epithelium, etc.),
muscle tissues (including skeletal muscles, cardiac muscles, smooth
muscles, etc.), urogenital tissues (e.g., kidney, pronephros, meta- and
meso-nephric ducts, metanephric diverticulum, ureters, renal pelvis,
collecting tubules, epithelium of the female reproductive structures
(particularly the oviducts, uterus, and vagina)), pleural and peritoneal
tissues, viscera, mesodermal glandular tissues (e.g., adrenal cortex
tissues), and stromal tissues (e.g., bone marrow). Of course, inasmuch as
the cell can retain potential to develop into mature cells, it also can
realize its developmental phenotypic potential by differentiating into an
appropriate precursor cell (e.g., a preadipocyte, a premyocyte, a
preosteocyte, etc.). Also, depending on the culture conditions, the cells
can also exhibit developmental phenotypes such as embryonic, fetal,
hematopoetic, neurogenic, or neuralgiagenic developmental phenotypes. In
this sense, the inventive cell can have two or more developmental
phenotypes such as adipogenic, chondrogenic, cardiogenic, dermatogenic,
hematopoetic, hemangiogenic, myogenic, nephrogenic, neurogenic,
neuralgiagenic, urogenitogenic, osteogenic, pericardiogenic,
peritoneogenic, pleurogenic, splanchogenic, and stromal developmental
phenotypes. While such cells can retain two or more of these
developmental phenotypes, preferably, such cells have three or more such
developmental phenotypes (e.g., four or more mesodermal or mesenchymal
developmental phenotypes), and some types of inventive stem cells have a
potential to acquire any mesodermal phenotype through the process of
differentiation.

[0007]The inventive stem cell can be obtained from adipose tissue by any
suitable method. A first step in any such method requires the isolation
of adipose tissue from the source animal. The animal can be alive or
dead, so long as adipose stromal cells within the animal are viable.
Typically, human adipose stromal cells are obtained from living donors,
using well-recognized protocols such as surgical or suction lipectomy.
Indeed, as liposuction procedures are so common, liposuction effluent is
a particularly preferred source from which the inventive cells can be
derived.

[0008]However derived, the adipose tissue is processed to separate stem
cells from the remainder of the material. In one protocol, the adipose
tissue is washed with physiologically-compatible saline solution (e.g.,
phosphate buffered saline (PBS)) and then vigorously agitated and left to
settle, a step that removes loose matter (e.g., damaged tissue, blood,
erythrocytes, etc.) from the adipose tissue. Thus, the washing and
settling steps generally are repeated until the supernatant is relatively
clear of debris.

[0009]The remaining cells generally will be present in lumps of various
size, and the protocol proceeds using steps gauged to degrade the gross
structure while minimizing damage to the cells themselves. One method of
achieving this end is to treat the washed lumps of cells with an enzyme
that weakens or destroys bonds between cells (e.g., collagenase, dispase,
trypsin, etc.). The amount and duration of such enzymatic treatment will
vary, depending on the conditions employed, but the use of such enzymes
is generally known in the art. Alternatively or in conjunction with such
enzymatic treatment, the lumps of cells can be degraded using other
treatments, such as mechanical agitation, sonic energy, thermal energy,
etc. If degradation is accomplished by enzymatic methods, it is desirable
to neutralize the enzyme following a suitable period, to minimize
deleterious effects on the cells.

[0010]The degradation step typically produces a slurry or suspension of
aggregated cells (generally liposomes) and a fluid fraction containing
generally free stromal cells (e.g., red blood cells, smooth muscle cells,
endothelial cells, fibroblast cells, and stem cells). The next stage in
the separation process is to separate the aggregated cells from the
stromal cells. This can be accomplished by centrifugation, which forces
the stromal cells into a pellet covered by a supernatant. The supernatant
then can be discarded and the pellet suspended in a
physiologically-compatible fluid. Moreover, the suspended cells typically
include erythrocytes, and in most protocols it is desirable to lyse
these. Methods for selectively lysing erythrocytes are known in the art,
and any suitable protocol can be employed (e.g., incubation in a hyper-
or hypotonic medium). Of course, if the erythrocytes are lysed, the
remaining cells should then be separated from the lysate, for example by
filtration or centrifugation. Of course, regardless of whether the
erythrocytes are lysed, the suspended cells can be washed,
re-centrifuged, and resuspended one or more successive times to achieve
greater purity. Alternatively, the cells can be separated using a cell
sorter or on the basis of cell size and granularity, stem cells being
relatively small and agranular. Expression of telomerase can also serve
as a stem cell-specific marker. They can also be separated
immunohistochemically, for example, by panning or using magnetic beads.
Any of the steps and procedures for isolating the inventive cells can be
performed manually, if desired. Alternatively, the process of isolating
such cells can be facilitated through a suitable device, many of which
are known in the art (see, e.g., U.S. Pat. No. 5,786,207).

[0011]Following the final isolation and resuspension, the cells can be
cultured and, if desired, assayed for number and viability to assess the
yield. Desirably the cells can be cultured without differentiation using
standard cell culture media (e.g., DMEM, typically supplemented with
5-15% (e.g., 10%) serum (e.g., fetal bovine serum, horse serum, etc.).
Preferably, the cells can be passaged at least five times in such medium
without differentiating, while still retaining their developmental
phenotype, and more preferably, the cells can be passaged at least 10
times (e.g., at least 15 times or even at least 20 times) without losing
developmental phenotype. Thus, culturing the cells of the present
invention without inducing differentiation can be accomplished without
specially screened lots of serum, as is generally the case for
mesenchymal stem cells (e.g., derived from marrow). Methods for measuring
viability and yield are known in the art (e.g., trypan blue exclusion).

[0012]Following isolation, the stem cells are further separated by
phenotypic identification, to identify those cells that have two or more
of the aforementioned developmental phenotypes. Typically, the stromal
cells are plated at a desired density such as between about 100
cells/cm2 to about 100,000 cells/cm2 (such as about 500
cells/cm2 to about 50,000 cells/cm2, or, more particularly,
between about 1,000 cells/cm2 to about 20,000 cells/cm2). If
plated at lower densities (e.g., about 300 cells/cm2), the cells can
be more easily clonally isolated. For example, after a few days, cells
plated at such densities will proliferate into a population.

[0013]Such cells and populations can be clonally expanded, if desired,
using a suitable method for cloning cell populations. For example, a
proliferated population of cells can be physically picked and seeded into
a separate plate (or the well of a multi-well plate). Alternatively, the
cells can be subcloned onto a multi-well plate at a statistical ratio for
facilitating placing a single cell into each well (e.g., from about 0.1
to about 1 cell/well or even about 0.25 to about 0.5 cells/well, such as
0.5 cells/well). Of course, the cells can be cloned by plating them at
low density (e.g., in a petri-dish or other suitable substrate) and
isolating them from other cells using devices such as a cloning rings.
Alternatively, where an irradiation source is available, clones can be
obtained by permitting the cells to grow into a monolayer and then
shielding one and irradiating the rest of cells within the monolayer. The
surviving cell then will grow into a clonal population. While production
of a clonal population can be expanded in any suitable culture medium, a
preferred culture condition for cloning stem cells (such as the inventive
stem cells or other stem cells) is about 2/3 F12 medium+20% serum
(preferably fetal bovine serum) and about 1/3 standard medium that haw
been conditioned with stromal cells (e.g., cells from the stromal
vascular fraction of liposuction aspirate), the relative proportions
being determined volumetrically).

[0014]In any event, whether clonal or not, the isolated cells can be
cultured to a suitable point when their developmental phenotype can be
assessed. As mentioned, the inventive cells have at least two of the
aforementioned developmental phenotypes. Thus, one or more cells drawn
from a given clone can be treated to ascertain whether it possesses such
developmental potentials. One type of treatment is to culture the
inventive cells in culture media that has been conditioned by exposure to
mature cells (pr precursors thereof) of the respective type to be
differentiated (e.g., media conditioned by exposure to myocytes can
induce myogenic differentiation, media conditioned by exposure to heart
valve cells can induce differentiation into heart valve tissue, etc.). Of
course, defined media for inducing differentiation also can be employed.
For example, adipogenic developmental phenotype can be assessed by
exposing the cell to a medium that facilitates adipogenesis, e.g.,
containing a glucocorticoid (e.g., isobutyl-methylxanthine,
dexamethasone, hydrocortisone, cortisone, etc.), insulin, a compound
which elevates intracellular levels of cAMP (e.g., dibutyryl-cAMP,
8-CPT-cAMP (8-(4)chlorophenylthio)-adenosine 3',5' cyclic monophosphate;
8-bromo-cAMP; dioctanoyl-cAMP, forskolin etc.), and/or a compound which
inhibits degradation of cAMP (e.g., a phosphodiesterase inhibitor such as
methyl isobutylxanthine, theophylline, caffeine, indomethacin, and the
like). Thus, exposure of the stem cells to between about 1 μM and
about 10 μM insulin in combination with about 10-9 M to about
10-6 M to (e.g., about 1 μM) dexamethasone can induce adipogenic
differentiation. Such a medium also can include other agents, such as
indomethicin (e.g., about 100 μM to about 200 μM), if desired, and
preferably the medium is serum free. Osteogenic developmental phenotype
can be assessed by exposing the cells to between about 10-7 M and
about 10-9 M dexamethasone (e.g., about 1 μM) in combination with
about 10 μM to about 50 μM ascorbate-2-phosphate and between about
10 nM and about 50 nM β-glycerophosphate, and the medium also can
include serum (e.g., bovine serum, horse serum, etc.). Myogenic
differentiation can be induced by exposing the cells to between about 10
μM and about 100 μM hydrocortisone, preferably in a serum-rich
medium (e.g., containing between about 10% and about 20% serum (either
bovine, horse, or a mixture thereof)). Chondrogenic differentiation can
be induced by exposing the cells to between about 1 μM to about 10
μM insulin and between about 1 μM to about 10 μM transferrin,
between about 1 ng/ml and 10 ng/ml transforming growth factor (TGF)
β1, and between about 10 nM and about 50 nM ascorbate-2-phosphate
(50 nM). For chondrogenic differentiation, preferably the cells are
cultured in high density (e.g., at about several million cells/ml or
using micromass culture techniques), and also in the presence of low
amounts of serum (e.g., from about 1% to about 5%). The cells also can be
induced to assume a developmentally more immature phenotype (e.g., a
fetal or embryonic phenotype). Such induction is achieved upon exposure
of the inventive cell to conditions that mimic those within fetuses and
embryos. For example, the inventive cells or populations can be
co-cultured with cells isolated from fetuses or embryos, or in the
presence of fetal serum. Along these lines, the cells can be induced to
differentiate into any of the aforementioned mesodermal lineages by
co-culturing them with mature cells of the respective type, or precursors
thereof. Thus, for example, myogenic differentiation can be induced by
culturing the inventive cells with myocytes or precursors, and similar
results can be achieved with respect to the other tissue types mentioned
herein. Other methods of inducing differentiation are known in the art,
and many of them can be employed, as appropriate.

[0015]After culturing the cells in the differentiating-inducing medium for
a suitable time (e.g., several days to a week or more), the cells can be
assayed to determine whether, in fact, they have differentiated to
acquire physical qualities of a given type of cell. One measurement of
differentiation per se is telomere length, undifferentiated stem cells
having longer telomeres than differentiated cells; thus the cells can be
assayed for the level of telomerase activity. Alternatively, RNA or
proteins can be extracted from the cells and assayed (via Northern
hybridization, rtPCR, Western blot analysis, etc.) for the presence of
markers indicative of the desired phenotype. Of course, the cells can be
assayed immunohistochemically or stained, using tissue-specific stains.
Thus, for example, to assess adipogenic differentiation, the cells can be
stained with fat-specific stains (e.g., oil red O, safarin red, sudan
black, etc.) or probed to assess the presence of adipose-related factors
(e.g., type IV collagen, PPAR-γ, adipsin, lipoprotein lipase,
etc.). Similarly, ostogenesis can be assessed by staining the cells with
bone-specific stains (e.g., alkaline phosphatase, von Kossa, etc.) or
probed for the presence of bone-specific markers (e.g., osteocalcin,
osteonectin, osteopontin, type I collagen, bone morphogenic proteins,
cbfa, etc.). Myogensis can be assessed by identifying classical
morphologic changes (e.g., polynucleated cells, syncitia formation,
etc.), or assessed biochemically for the presence of muscle-specific
factors (e.g., myo D, myosin heavy chain, NCAM, etc.). Chondrogenesis can
be determined by staining the cells using cartallge-specific stains
(e.g., alcian blue) or probing the cells for the expression/production of
cartilage-specific molecules (e.g., sulfated glycosaminoglycans and
proteoglycans (e.g., keratin, chondroitin, etc.) in the medium, type II
collagen, etc.). Other methods of assessing developmental phenotype are
known in the art, and any of them is appropriate. For example, the cells
can be sorted by size and granularity. Also, the cells can be used to
generate monoclonal antibodies, which can then be employed to assess
whether they preferentially bind to a given cell type. Correlation of
antigenicity can confirm that the stem cell has differentiated along a
given developmental pathway.

[0016]While the cell can be solitary and isolated from other cells,
preferably it is within a population of cells, and the invention provides
a defined population including the inventive cell. In some embodiments,
the population is heterogeneous. Thus, for example, the population can
include support cells for supplying factors to the inventive cells. Of
course, the inventive stem cells can themselves serve as support cells
for culturing other types of cells (such as other types of stem cells,
e.g., as neural stem cells (NSC), hematopoetic stem cells (HPC,
particularly CD34.sup.+ stem cells), embryonic stem cells (ESC) and
mixtures thereof), and the population can include such cells. In other
embodiments, the population is substantially homogeneous, consisting
essentially of the inventive lipo-derived stem cells.

[0017]As the inventive cells can be cloned, a substantially homogeneous
population containing them can be clonal. Indeed, the invention also
pertains to any defined clonal cell population consisting essentially of
mesodermal stem cells, connective tissue stem cell, or mixtures thereof.
In this embodiment, the cells can be lipo-derived or derived from other
mesodermal or connective cell tissues (e.g., bone marrow, muscle, etc.)
using methods known in the art. After the isolation, the cells can be
expanded clonally as described herein.

[0018]The inventive cells (and cell populations) can be employed for a
variety of purposes. As mentioned, the cells can support the growth and
expansion of other cell types, and the invention pertains to methods for
accomplishing this. In one aspect, the invention pertains to a method of
conditioning culture medium using the inventive stem cells and to
conditioned medium produced by such a method. The medium becomes
conditioned upon exposing a desired culture medium to the cells under
conditions sufficient for the cells to condition it. Typically, the
medium is used to support the growth of the inventive cells, which
secrete hormones, cell matrix material, and other factors into the
medium. After a suitable period (e.g., one or a few days), the culture
medium containing the secreted factors can be separated from the cells
and stored for future use. Of course, the inventive cells and populations
can be re-used successively to condition medium, as desired. In other
applications (e.g., for co-culturing the inventive cells with other cell
types), the cells can remain within the conditioned medium. Thus, the
invention provides a conditioned medium obtained using this method, which
either can contain the inventive cells or be substantially free of the
inventive cells, as desired.

[0019]The conditioned medium can be used to support the growth and
expansion of desired cell types, and the invention provides a method of
culturing cells (particularly stem cells) using the conditioned medium.
The method involves maintaining a desired cell in the conditioned medium
under conditions for the cell to remain viable. The cell can be
maintained under any suitable condition for culturing them, such as are
known in the art. Desirably, the method permits successive rounds of
mitotic division of the cell to form an expanded population. The exact
conditions (e.g., temperature, CO2 levels, agitation, presence of
antibiotics, etc.) will depend on the other constituents of the medium
and on the cell type. However, optimizing these parameters are within the
ordinary skill in the art. In some embodiments, it is desirable for the
medium to be substantially free of the lipo-derived cells employed to
condition the medium as described herein. However, in other embodiments,
it is desirable for the lipo-derived cells to remain in the conditioned
medium and co-cultured with the cells of interest. Indeed, as the
inventive lipo-derived cells can express cell-surface mediators of
intercellular communication, it often is desirable for the inventive
cells and the desired other cells to be co-cultured under conditions in
which the two cell types are in contact. This can be achieved, for
example, by seeding the cells as a heterogeneous population of cells onto
a suitable culture substrate. Alternatively, the inventive lipo-derived
cells can first be grown to confluence, which will serve as a substrate
for the second desired cells to be cultured within the conditioned
medium.

[0020]In another embodiment, the inventive lipo-derived cells can be
genetically modified, e.g., to express exogenous genes or to repress the
expression of endogenous genes, and the invention provides a method of
genetically modifying such cells and populations. In accordance with this
method, the cell is exposed to a gene transfer vector comprising a
nucleic acid including a transgene, such that the nucleic acid is
introduced into the cell under conditions appropriate for the transgene
to be expressed within the cell. The transgene generally is an expression
cassette, including a coding polynucleotide operably linked to a suitable
promoter. The coding polynucleotide can encode a protein, or it can
encode biologically active RNA (e.g., antisense RNA or a ribozyme). Thus,
for example, the coding polynucleotide can encode a gene conferring
resistance to a toxin, a hormone (such as peptide growth hormones,
hormone releasing factors, sex hormones, adrenocorticotrophic hormones,
cytokines (e.g., interferins, interleukins, lymphokines), etc.), a
cell-surface-bound intracellular signaling moiety (e.g., cell adhesion
molecules, hormone receptors, etc.), a factor promoting a given lineage
of differentiation, etc. Of course, where it is desired to employ gene
transfer technology to deliver a given transgene, its sequence will be
known.

[0021]Within the expression cassette, the coding polynucleotide is
operably linked to a suitable promoter. Examples of suitable promoters
include prokaryotic promoters and viral promoters (e.g., retroviral ITRs,
LTRs, immediate early viral promoters (IEp), such as herpesvirus IEp
(e.g., ICP4-IEp and ICP0-IEp), cytomegalovirus (CMV) IEp, and other viral
promoters, such as Rous Sarcoma Virus (RSV) promoters, and Murine
Leukemia Virus (MLV) promoters). Other suitable promoters are eukaryotic
promoters, such as enhancers (e.g., the rabbit β-globin regulatory
elements), constitutively active promoters (e.g., the β-actin
promoter, etc.), signal specific promoters (e.g., inducible promoters
such as a promoter responsive to RU486, etc.), and tissue-specific
promoters. It is well within the skill of the art to select a promoter
suitable for driving gene expression in a predefined cellular context.
The expression cassette can include more than one coding polynucleotide,
and it can include other elements (e.g., polyadenylation sequences,
sequences encoding a membrane-insertion signal or a secretion leader,
ribosome entry sequences, transcriptional regulatory elements (e.g.,
enhancers, silencers, etc.), and the like), as desired.

[0022]The expression cassette containing the transgene should be
incorporated into a genetic vector suitable for delivering the transgene
to the cells. Depending on the desired end application, any such vector
can be so employed to genetically modify the cells (e.g., plasmids, naked
DNA, viruses such as adenovirus, adeno-associated virus, herpesviruses,
lentiviruses, papillomaviruses, retroviruses, etc.). Any method of
constructing the desired expression cassette within such vectors can be
employed, many of which are well known in the art (e.g., direct cloning,
homologous recombination, etc.). Of course, the choice of vector will
largely determine the method used to introduce the vector into the cells
(e.g., by protoplast fusion, calcium-phosphate precipitation, gene gun,
electroporation, infection with viral vectors, etc.), which are generally
known in the art.

[0023]The genetically altered cells can be employed as bioreactors for
producing the product of the transgene. In other embodiments, the
genetically modified cells are employed to deliver the transgene and its
product to an animal. For example, the cells, once genetically modified,
can be introduced into the animal under conditions sufficient for the
transgene to be expressed in vivo.

[0024]In addition to serving as useful targets for genetic modification,
many cells and populations of the present invention secrete hormones
(e.g., cytokines, peptide or other (e.g., monobutyrin) growth factors,
etc.). Some of the cells naturally secrete such hormones upon initial
isolation, and other cells can be genetically modified to secrete
hormones, as discussed herein. The cells of the present invention that
secrete hormones can be used in a variety of contexts in vivo and in
vitro. For example, such cells can be employed as bioreactors to provide
a ready source of a given hormone, and the invention pertains to a method
of obtaining hormones from such cells. In accordance with the method, the
cells are cultured, under suitable conditions for them to secrete the
hormone into the culture medium. After a suitable period of time, and
preferably periodically, the medium is harvested and processed to isolate
the hormone from the medium. Any standard method (e.g., gel or affinity
chromatography, dialysis, lyophilization, etc.) can be used to purify the
hormone from the medium, many of which are known in the art.

[0025]In other embodiments, cells (and populations) of the present
invention secreting hormones can be employed as therapeutic agents.
Generally, such methods involve transferring the cells to desired tissue,
either in vitro (e.g., as a graft prior to implantation or engrafting) or
in vivo, to animal tissue directly. The cells can be transferred to the
desired tissue by any method appropriate, which generally will vary
according to the tissue type. For example, cells can be transferred to a
graft by bathing the graft (or infusing it) with culture medium
continuing the cells. Alternatively, the cells can be seeded onto the
desired site within the tissue to establish a population. Cells can be
transferred to sites in vivo using devices such as catheters, trocars,
cannulae, stents (which can be seeded with the cells), etc. For these
applications, preferably the cell secretes a cytokine or growth hormone
such as human growth factor, fibroblast growth factor, nerve growth
factor, insulin-like growth factors, hemopoietic stem cell growth
factors, members of the fibroblast growth factor family, members of the
platelet-derived growth factor family, vascular and endothelial cell
growth factors, members of the TGFb family (including bone morphogenic
factor), or enzymes specific for congenital disorders (e.g., distrophin).

[0026]In one application, the invention provides a method of promoting the
closure of a wound within a patient using such cells. In accordance with
the method, the inventive cells secreting the hormone are transferred to
the vicinity of a wound under conditions sufficient for the cell to
produce the hormone. The presence of the hormone in the vicinity of the
wound promotes closure of the wound. The method promotes closure of both
external (e.g., surface) and internal wounds. Wounds to which the present
inventive method is useful in promoting closure include, but are not
limited to, abrasions, avulsions, blowing wounds, burn wounds,
contusions, gunshot wounds, incised wounds, open wounds, penetrating
wounds, perforating wounds, puncture wounds, seton wounds, stab wounds,
surgical wounds, subcutaneous wounds, or tangential wounds. The method
need not achieve complete healing or closure of the wound; it is
sufficient for the method to promote any degree of wound closure. In this
respect, the method can be employed alone or as an adjunct to other
methods for healing wounded tissue.

[0027]Where the inventive cells secrete an angiogenic hormone (e.g.,
vascular growth factor, vascular and endothelial cell growth factor,
etc.), they (as well as populations containing them) can be employed to
induce angiogenesis within tissues. Thus, the invention provides a method
of promoting neovascularization within tissue using such cells. In
accordance with this method, the cell is introduced the desired tissue
under conditions sufficient for the cell to produce the angiogenic
hormone. The presence of the hormone within the tissue promotes
neovascularization within the tissue.

[0028]Because the inventive stem cells have a developmental phenotype,
they can be employed in tissue engineering. In this regard, the invention
provides a method of producing animal matter comprising maintaining the
inventive cells under conditions sufficient for them to expand and
differentiate to form the desired matter. The matter can include mature
tissues, or even whole organs, including tissue types into which the
inventive cells can differentiate (as set forth herein). Typically, such
matter will comprise adipose, cartilage, heart, dermal connective tissue,
blood tissue, muscle, kidney, bone, pleural, splancnic tissues, vascular
tissues, and the like. More typically, the matter will comprise
combinations of these tissue types (i.e., more than one tissue type). For
example, the matter can comprise all or a portion of an animal organ
(e.g., a heart, a kidney) or a limb (e.g., a leg, a wing, an arm, a hand,
a foot, etc.). Of course, in as much as the cells can divide and
differentiate to produce such structures, they can also form anlagen of
such structures. At early stages, such anlagen can be cryopreserved for
future generation of the desired mature structure or organ.

[0029]To produce such structures, the inventive cells and populations are
maintained under conditions suitable for them to expand and divide to
form the desired structures. In some applications, this is accomplished
by transferring them to an animal (i.e., in vivo) typically at a sight at
which the new matter is desired. Thus, for example, the invention can
facilitate the regeneration of tissues (e.g., bone, muscle, cartilage,
tendons, adipose, etc.) within an animal where the cells are implanted
into such tissues. In other embodiments, and particularly to create
anlagen, the cells can be induced to differentiate and expand into
tissues in vitro. In such applications, the cells are cultured on
substrates that facilitate formation into three-dimensional structures
conducive for tissue development. Thus, for example, the cells can be
cultured or seeded onto a bio-compatible lattice, such as one that
includes extracellular matrix material, synthetic polymers, cytokines,
growth factors, etc. Such a lattice can be molded into desired shapes for
facilitating the development of tissue types. Also, at least at an early
stage during such culturing, the medium and/or substrate is supplemented
with factors (e.g., growth factors, cytokines, extracellular matrix
material, etc.) that facilitate the development of appropriate tissue
types and structures. Indeed, in some embodiments, it is desired to
co-culture the cells with mature cells of the respective tissue type, or
precursors thereof, or to expose the cells to the respective conditioned
medium, as discussed herein.

[0030]To facilitate the use of the inventive lipo-derived cells and
populations for producing such animal matter and tissues, the invention
provides a composition including the inventive cells (and populations)
and biologically compatible lattice. Typically, the lattice is formed
from polymeric material, having fibers as a mesh or sponge, typically
with spaces on the order of between about 100 μm and about 300 μm.
Such a structure provides sufficient area on which the cells can grow and
proliferate. Desirably, the lattice is biodegradable over time, so that
it will be absorbed into the animal matter as it develops. Suitable
polymeric lattices, thus, can be formed from monomers such as glycolic
acid, lactic acid, propyl fumarate, caprolactone, hyaluronan, hyaluronic
acid, and the like. Other lattices can include proteins, polysaccharides,
polyhydroxy acids, polyorthoesters, polyanhydrides, polyphosphazenes, or
synthetic polymers (particularly biodegradable polymers). Of course, a
suitable polymer for forming such lattice can include more than one
monomers (e.g., combinations of the indicated monomers). Also, the
lattice can also include hormones, such as growth factors, cytokines, and
morphogens (e.g., retinoic acid, aracadonic acid, etc.), desired
extracellular matrix molecules (e.g., fibronectin, laminin, collagen,
etc.), or other materials (e.g., DNA, viruses, other cell types, etc.) as
desired.

[0031]To form the composition, the cells are introduced into the lattice
such that they permeate into the interstitial spaces therein. For
example, the matrix can be soaked in a solution or suspension containing
the cells, or they can be infused or injected into the matrix. A
particularly preferred composition is a hydrogel formed by crosslinking
of a suspension including the polymer and also having the inventive cells
dispersed therein. This method of formation permits the cells to be
dispersed throughout the lattice, facilitating more even permeation of
the lattice with the cells. Of course, the composition also can include
mature cells of a desired phenotype or precursors thereof, particularly
to potentate the induction of the inventive stem cells to differentiate
appropriately within the lattice (e.g., as an effect of co-culturing such
cells within the lattice).

[0032]The composition can be employed in any suitable manner to facilitate
the growth and generation of the desired tissue types, structures, or
anlagen. For example, the composition can be constructed using
three-dimensional or stereotactic modeling techniques. Thus, for example,
a layer or domain within the composition can be populated by cells primed
for osteogenic differentiation, and another layer or domain within the
composition can be populated with cells primed for myogenic and/or
chondrogenic development. Bringing such domains into juxtaposition with
each other facilitates the molding and differentiation of complex
structures including multiple tissue types (e.g., bone surrounded by
muscle, such as found in a limb). To direct the growth and
differentiation of the desired structure, the composition can be cultured
ex vivo in a bioreactor or incubator, as appropriate. In other
embodiments, the structure is implanted within the host animal directly
at the site in which it is desired to grow the tissue or structure. In
still another embodiment, the composition can be engrafted on a host
(typically an animal such as a pig, baboon, etc.), where it will grow and
mature until ready for use. Thereafter, the mature structure (or anlage)
is excised from the host and implanted into the host, as appropriate.

[0033]Lattices suitable for inclusion into the composition can be derived
from any suitable source (e.g., matrigel), and some commercial sources
for suitable lattices exist (e.g., suitable of polyglycolic acid can be
obtained from sources such as Ethicon, N.J.). Another suitable lattice
can be derived from the acellular portion of adipose tissue--i.e.,
adipose tissue extracellular matrix matter substantially devoid of cells,
and the invention provides such a lipo-derived lattice. Typically, such
lipo-derived lattice includes proteins such as proteoglycans,
glycoproteins, hyaluronins, fibronectins, collagens (type I, type II,
type III, type IV, type V, type VI, etc.), and the like, which serve as
excellent substrates for cell growth. Additionally, such lipo-derived
lattices can include hormones, preferably cytokines and growth factors,
for facilitating the growth of cells seeded into the matrix.

[0034]The lipo-derived matrix can be isolated form adipose tissue
similarly as described above, except that it will be present in the
acellular fraction. For example, adipose tissue or derivatives thereof
(e.g., a fraction of the cells following the centrifugation as discussed
above) can be subjected to sonic or thermal energy and/or enzymatic
processed to recover the matrix material. Also, desirably the cellular
fraction of the adipose tissue is disrupted, for example by treating it
with lipases, detergents, proteases, and/or by mechanical or sonic
disruption (e.g., using a homogenizer or sonicator). However isolated,
the material is initially identified as a viscous material, but it can be
subsequently treated, as desired, depending on the desired end use. For
example, the raw matrix material can be treated (e.g., dialyzed or
treated with proteases or acids, etc.) to produce a desirable lattice
material. Thus the lattice can be prepared in a hydrated form or it can
be dried or lyophilized into a substantially anhydrous form or a powder.
Thereafter, the powder can be rehydrated for use as a cell culture
substrate, for example by suspending it in a suitable cell culture
medium. In this regard, the lipo-derived lattice can be mixed with other
suitable lattice materials, such as described above. Of course, the
invention pertains to compositions including the lipo-derived lattice and
cells or populations of cells, such as the inventive lipo-derived cells
and other cells as well (particularly other types of stem cells).

[0035]As discussed above, the cells, populations, lattices, and
compositions of the invention can be used in tissue engineering and
regeneration. Thus, the invention pertains to an implantable structure
(i.e., an implant) incorporating any of these inventive features. The
exact nature of the implant will vary according to the use to which it is
to be put. The implant can be or comprise, as described, mature tissue,
or it can include immature tissue or the lattice. Thus, for example, one
type of implant can be a bone implant, comprising a population of the
inventive cells that are undergoing (or are primed for) osteogenic
differentiation, optionally seeded within a lattice of a suitable size
and dimension, as described above. Such an implant can be injected or
engrafted within a host to encourage the generation or regeneration of
mature bone tissue within the patient. Similar implants can be used to
encourage the growth or regeneration of muscle, fat, cartilage, tendons,
etc., within patients. Other types of implants are anlagen (such as
described herein), e.g., limb buds, digit buds, developing kidneys, etc,
that, once engrafted onto a patient, will mature into the appropriate
structures.

[0036]The lipo-derived lattice can conveniently be employed as part of a
cell culture kit. Accordingly, the invention provides a kit including the
inventive lipo-derived lattice and one or more other components, such as
hydrating agents (e.g., water, physiologically-compatible saline
solutions, prepared cell culture media, serum or derivatives thereof,
etc.), cell culture substrates (e.g., culture dishes, plates, vials,
etc.), cell culture media (whether in liquid or powdered form),
antibiotic compounds, hormones, and the like. While the kit can include
any such ingredients, preferably it includes all ingredients necessary to
support the culture and growth of desired cell types upon proper
combination. Of course, if desired, the kit also can include cells
(typically frozen), which can be seeded into the lattice as described
herein.

[0037]While many aspects of the invention pertain to tissue growth and
differentiation, the invention has other applications as well. For
example, the lipo-derived lattice can be used as an experimental reagent,
such as in developing improved lattices and substrates for tissue growth
and differentiation. The lipo-derived lattice also can be employed
cosmetically, for example, to hide wrinkles, scars, cutaneous
depressions, etc., or for tissue augmentation. For such applications,
preferably the lattice is stylized and packaged in unit dosage form. If
desired, it can be admixed with carriers (e.g., solvents such as
glycerine or alcohols), perfumes, antibiotics, colorants, and other
ingredients commonly employed in cosmetic products. The substrate also
can be employed autologously or as an allograft, and it can used as, or
included within, ointments or dressings for facilitating wound healing.
The lipo-derived cells can also be used as experimental reagents. For
example, they can be employed to help discover agents responsible for
early events in differentiation. For example, the inventive cells can be
exposed to a medium for inducing a particular line of differentiation and
then assayed for differential expression of genes (e.g., by random-primed
PCR or electrophoresis or protein or RNA, etc.).

[0038]As any of the steps for isolating the inventive stem cells or the
lipo-derived lattice, the, the invention provides a kit for isolating
such reagents from adipose tissues. The kit can include a means for
isolating adipose tissue from a patient (e.g., a cannula, a needle, an
aspirator, etc.), as well as a means for separating stromal cells (e.g.,
through methods described herein). The kit can be employed, for example,
as a bedside source of stem cells that can then be re-introduced from the
same individual as appropriate. Thus, the kit can facilitate the
isolation of lipo-derived stem cells for implantation in a patient
needing regrowth of a desired tissue type, even in the same procedure. In
this respect, the kit can also include a medium for differentiating the
cells, such as those set forth herein. As appropriate, the cells can be
exposed to the medium to prime them for differentiation within the
patient as needed. Of course, the kit can be used as a convenient source
of stem cells for in vitro manipulation (e.g., cloning or differentiating
as described herein). In another embodiment, the kit can be employed for
isolating a lipo-derived lattice as described herein.

EXAMPLES

[0039]While one of skill in the art is fully able to practice the instant
invention upon reading the foregoing detailed description, the following
examples will help elucidate some of its features. In particular, they
demonstrate the isolation of a human lipo-derived stem cell substantially
free of mature adipocytes, the isolation of a clonal population of such
cells, the ability of such cells to differentiate in vivo and in vitro,
and the capacity of such cells to support the growth of other types of
stem cells. The examples also demonstrate the isolation of a lipo-derived
lattice substantially free of cells that is capable of serving as a
suitable substrate for cell culture. Of course, as these examples are
presented for purely illustrative purposes, they should not be used to
construe the scope of the invention in a limited manner, but rather
should be seen as expanding upon the foregoing description of the
invention as a whole.

[0040]The procedures employed in these examples, such as surgery, cell
culture, enzymatic digestion, histology, and molecular analysis of
proteins and polynucleotides, are familiar to those of ordinary skill in
this art. As such, and in the interest of brevity, experimental details
are not recited in detail.

[0042]Raw liposuction aspirate was obtained from patients undergoing
elective surgery. Prior to the liposuction procedures, the patients were
given epinephrine to minimize contamination of the aspirate with blood.
The aspirate was strained to separate associated adipose tissue pieces
from associated liquid waste. Isolated tissue was rinsed thoroughly with
neutral phosphate buffered saline and then enzymatically dissociated with
0.075% w/v collagenase at 37° C. for about 20 minutes under
intermittent agitation. Following the digestion, the collagenase was
neutralized, and the slurry was centrifuged at about 260 g for about 10
minutes, which produced a multi-layered supernatant and a cellular
pellet. The supernatant was removed and retained for further use, and the
pellet was resuspended in an erythrocyte-lysing solution and incubated
without agitation at about 25° C. for about 10 minutes. Following
incubation, the medium was neutralized, and the cells were again
centrifuged at about 250 g for about 10 minutes. Following the second
centrifugation, the cells were suspended, and assessed for viability
(using trypan blue exclusion) and cell number. Thereafter, they were
plated at a density of about at about 1×106 cells/100 mm dish.
They were cultured at 37° C. in DMEM+fetal bovine serum (about
10%) in about 5% CO2.

[0043]The majority of the cells were adherent, small, mononucleic,
relatively agranular fibroblast-like cells containing no visible lipid
droplets. The majority the cells stained negatively with oil-red O and
von Kossa. The cells were also assayed for expression of telomerase
(using a commercially available TRAP assay kit), using HeLa cells and
HN-12 cells as positive controls. Human foreskin fibroblasts and HN-12
heated cell extracts were used as negative controls. Telomeric products
were resolved onto a 12.5% polyacrylamide cells and the signals
determined by phosphorimaging. Telemeric ladders representing telomerase
activity were observed in the adipose-derived stem cells as well as the
positive controls. No ladders were observed in the negative controls.

[0044]Thus, these cells were not identifiable as myocytes, adipocytes,
chondrocytes, osteocytes, or blood cells. These results demonstrate that
the adipose-derived cells express telomerase activity similar to that
previously reported for human stem cells.

[0045]Subpopulations of these cells were then exposed to the following
media to assess their developmental phenotype:

[0046]A population was cultured at high density in the chondrogenic medium
for several weeks. Histological analysis of the tissue culture and
paraffin sections was performed with H&E, alcian blue, toludene blue, and
Goldner's trichrome staining at 2, 7, and 14 days. Immunohistochemistry
was performed using antibodies against chondroitin-4-sulfate and keratin
sulfate and type II collagen. Qualitative estimate of matrix staining was
also performed. The results indicated that cartilaginous spheroid nodules
with a distinct border of perichondral cells formed as early as 48 hours
after initial treatment. Untreated control cells exhibited no evidence of
chondrogenic differentiation. These results confirm that the stem cells
have chondrogenic developmental phenotype.

[0047]A population was cultured until near confluence and then exposed to
the adipogenic medium for several weeks. The population was examined at
two and four weeks after plating by colorimetric assessment of relative
opacity following oil red-O staining. Adipogenesis was determined to be
underway at two weeks and quite advanced at four weeks (relative opacity
of 1 and 5.3, respectively). Bone marrow-derived stem cells were employed
as a positive control, and these cells exhibited slightly less adipogenic
potential (relative density of 0.7 and 2.8, respectively).

[0048]A population was cultured until near confluence and then exposed to
the osteogenic medium for several weeks. The population was examined at
two and four weeks after plating by colorimetric assessment of relative
opacity following von Kossa staining. Osteogenesis was determined to be
underway at two weeks and quite advanced at four weeks (relative opacity
of 1.1 and 7.3, respectively. Bone marrow-derived stem cells were
employed as a positive control, and these cells exhibited slightly less
osteogenic potential (relative density of 0.2 and 6.6, respectively).

[0049]A population was cultured until near confluence and then exposed to
the myogenic medium for several weeks. The population was examined at
one, three, and six weeks after plating by assessment of multinucleated
cells and expression of muscle-specific proteins (MyoD and myosin heavy
chain). Human foreskin fibroblasts and skeletal myoblasts were used as
controls. Cells expressing MyoD and myosin were found at all time points
following exposure to the myogenic medium in the stem cell population,
and the proportion of such cells increased at 3 and 6 weeks.
Multinucleated cells were observed at 6 weeks. In contrast, the
fibroblasts exhibited none of these characteristics at any time points.

[0051]This example demonstrates that lipo-derived stem cells do not
differentiate in response to 5-azacytidine.

[0052]Lipo-derived stem cells obtained in accordance with Example 1 were
cultured in the presence of 5-azacytidine. In contrast with bone
marrow-derived stem cells, exposure to this agent did not induce myogenic
differentiation (see Wakitani et al., supra).

Example 3

[0053]This example demonstrates the generation of a clonal population of
human lipo-derived stem cells.

[0054]Cells isolated in accordance with the procedure set forth in Example
1 were plated at about 5,000 cells/100 mm dish and cultured for a few
days as indicated in Example 1. After some rounds of cell division, some
clones were picked with a cloning ring and transferred to wells in a 48
well plate. These cells were cultured for several weeks, changing the
medium twice weekly, until they were about 80% to about 90% confluent (at
37° C. in about 5% CO2 in 2/3 F12 medium+20% fetal
bovine serum and 1/3 standard medium that was first conditioned by the
cells isolated in Example 1, "cloning medium"). Thereafter, each culture
was transferred to a 35 mm dish and grown, and then retransferred to a
100 mm dish and grown until close to confluent. Following this, one cell
population was frozen, and the remaining populations were plated on 12
well plates, at 1000 cells/well.

[0055]The cells were cultured for more than 15 passages in cloning medium
and monitored for differentiation as indicated in Example 1. The
undifferentiated state of each clone remained true after successive
rounds of differentiation.

[0056]Populations of the clones then were established and exposed to
adipogenic, chondrogenic, myogenic, and osteogenic medium as discussed in
Example 1. It was observed that at least one of the clones was able to
differentiate into bone, fat, cartilage, and muscle when exposed to the
respective media, and most of the clones were able to differentiate into
at least three types of tissues. The capacity of the cells to develop
into muscle and cartilage further demonstrates the pluripotentiality of
these lipo-derived stem cells.

[0057]These results demonstrate that the lipo-derived stem cells can be
maintained in an undifferentiated state for many passages without the
requirement for specially pre-screened lots of serum. The results also
demonstrate that the cells retain pluripotentiality following such
extensive passaging, proving that the cells are indeed stem cells and not
merely committed progenitor cells.

Example 4

[0058]This example demonstrates the lipo-derived stem cells can support
the culture of other types of stem cells.

[0059]Human lipo-derived stem cells were passaged onto 96 well plates at a
density of about 30,000/well, cultured for one week and then irradiated.
Human CD34.sup.+ hematopoetic stem cells isolated from umbilical cord
blood were then seeded into the wells. Co-cultures were maintained in
MyeloCult H5100 media, and cell viability and proliferation were
monitored subjectively by microscopic observation. After two weeks of
co-culture, the hematopoetic stem cells were evaluated for CD34
expression by flow cytometry.

[0060]Over a two-week period of co-culture with stromal cells, the
hematopoetic stem cells formed large colonies of rounded cells. Flow
analysis revealed that 62% of the cells remained CD34.sup.+. Based on
microscopic observations, human adipo-derived stromal cells maintained
the survival and supported the growth of human hematopoetic stem cells
derived from umbilical cord blood.

[0061]These results demonstrate that stromal cells from human subcutaneous
adipose tissue are able to support the ex vivo maintenance, growth and
differentiation of other stem cells.

Example 5

[0062]This example demonstrates that the lipo-derived stem cells can
differentiate in vivo.

[0063]Four groups (A-D) of 12 athymic mice each were implanted
subcutaneously with hydroxyapatite/tricalcium phosphate cubes containing
the following: Group A contained lipo-derived stem cells that had been
pretreated with osteogenic medium as set forth in Example 1. Group B
contained untreated lipo-derived stem cells. Group C contained osteogenic
medium but no cells. Group D contained non-osteogenic medium and no
cells. Within each group, six mice were sacrificed at three weeks, and
the remaining mice sacrificed at eight weeks following implantation. The
cubes were extracted, fixed, decalcified, and sectioned. Each section was
analyzed by staining with H&E, Mallory bone stain, and immunostaining for
osteocalcin.

[0064]Distinct regions of osteoid-like tissue staining for osteocalcin and
Mallory bone staining was observed in sections from groups A and B.
Substantially more osteoid tissue was observed in groups A and B than in
the other groups (p<0.05 ANOVA), but no significant difference in
osteogenesis was observed between groups A and B. Moreover, a qualitative
increase in bone growth was noted in both groups A and B between 3 and 8
weeks. These results demonstrate that the lipo-derived stem cells can
differentiate in vivo.

Example 6

[0065]This example demonstrates the isolation of a lipo-derived lattice
substantially devoid of cells.

[0066]In one protocol withheld supernatant from Example 1 was subjected to
enzymatic digestion for three days in 0.05% trypsin EDTA/100 U/ml
deoxyribonuclease to destroy the cells. Every day the debris was rinsed
in saline and fresh enzyme was added. Thereafter the material was rinsed
in saline and resuspended in 0.05% collagease and about 0.1% lipase to
partially digest the proteins and fat present. This incubation continued
for two days.

[0068]After both preparatory protocols, remaining substance was washed and
identified as a gelatinous mass. Microscopic analysis of this material
revealed that it contained no cells, and it was composed of high amounts
of collagen (likely type IV) and a wide variety of growth factors.
Preparations of this material have supported the growth of cells,
demonstrating it to be an excellent substrate for tissue culture.

INCORPORATION BY REFERENCE

[0069]All sources (e.g., inventor's certificates, patent applications,
patents, printed publications, repository accessions or records, utility
models, world-wide web pages, and the like) referred to or cited anywhere
in this document or in any drawing, Sequence Listing, or Statement filed
concurrently herewith are hereby incorporated into and made part of this
specification by such reference thereto.

GUIDE TO INTERPRETATION

[0070]The foregoing is an integrated description of the invention as a
whole, not merely of any particular element of facet thereof. The
description describes "preferred embodiments" of this invention,
including the best mode known to the inventors for carrying it out. Of
course, upon reading the foregoing description, variations of those
preferred embodiments will become obvious to those of ordinary skill in
the art. The inventors expect skilled artisans to employ such variations
as appropriate, and the inventors intend for the invention to be
practiced otherwise than as specifically described herein. Accordingly,
this invention includes all modifications and equivalents of the subject
matter recited in the claims appended hereto as permitted by applicable
law.

[0071]As used in the foregoing description and in the following claims,
singular indicators (e.g., "a" or "one") include the plural, unless
otherwise indicated. Recitation of a range of discontinuous values is
intended to serve as a shorthand method of referring individually to each
separate value falling within the range, and each separate value is
incorporated into the specification as if it were individually listed.
Additionally, the following terms are defined as follows: [0072]An
anlage is a primordial structure that has a capacity to develop into a
specific mature structure. [0073]A developmental phenotype is the
potential of a cell to acquire a particular physical phenotype through
the process of differentiation. [0074]A hormone is any substance that is
secreted by a cell and that causes a phenotypic change in the same or
another cell upon contact. [0075]A stem cell is a pluripotent cell that
has the capacity to differentiate in accordance with at least two
discrete developmental pathways.

[0076]As regards the claims in particular, the term "consisting
essentially of" indicates that unlisted ingredients or steps that do not
materially affect the basic and novel properties of the invention can be
employed in addition to the specifically recited ingredients or steps. In
contrast, terms such as "comprising," "having," and "including" indicate
that any ingredients or steps can be present in addition to those
recited. The term "consisting of" indicates that only the recited
ingredients or steps are present, but does not foreclose the possibility
that equivalents of the ingredients or steps can substitute for those
specifically recited.